Candida Infection Biology – fungal armoury, battlefields ... - FINSysB

Poster number: 27
C. glabrata cell wall attached and intracellular proteinases
Pirjo Pärnänen 1,2 , Pirjo Nikula-Ijäs 1
1 Department of Biosciences, Division of Biochemistry, University of Helsinki, Finland;
2 Institute of Dentistry, University of Helsinki, Finland
Background: C. glabrata is the most common non-albicans Candida species
causing oral mucosal infections and the third most common microbe that causes
invasive mortal hospital infections. C. glabrata has an an innate immunity to the
most common antifungal group azoles. Proteinases cause tissue protein
degradation and are involved in Candida virulence and invasion. Compared to the
most studied C. albicans, C. glabrata proteinases are mostly unfound or
uncharacterized. Against this background we studied clinical C. glabrata species
to locate and characterize these proteinases.
Materials and methods: Eight C. glabrata clinical strains, both mucosal and blood
isolates from hospitalized patients,were included. Gelatin zymography was used
for preliminary scanning of potential active proteinases and their inhibition by
proteinase inhibitors. The most gelatinolytic blood isolate (T-1639) was used for
further studies. Cell wall bound proteinases were enzymatically released and yeast
cells were sonicated to isolate plasma membrane and intracellular fractions in a
native state. Further activity testing was performed by fluorometric detection of
degraded synthetic L-Arg-AMC proteinase substrate.
Results: All the strains showed variable gelatinolytic activity by zymography. The
strain T-1639 showed enzymatic activity of approximate 25 kDa size released from
the 10x concentrated cell wall fraction, and more pronounced intracellular activity
of approximately 75 kDa size, which were both verified with fluorometry. The cell
wall bound proteinase was inhibited by a serine proteinase inhibitor (PMSF),and
the intracellular proteinase by a metalloproteinase inhibitor (EDTA).
Conclusions: The found proteinases are potentially new to our knowledge and
merrit further studies. The found proteinases belong to two different proteinase
classes and most probably have distinct roles in protein degradation and
pathogenic properties of C. glabrata.
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